DESCRIPTION: Heme proteins are ideally suited for detailed structure-function studies using protein engineering techniques. Most well studied heme proteins (globins, c-and b-type cytochromes, peroxidases, P450) all have exactly the same heme prosthetic group yet each exhibits very different and very clearly defined functions. These differences are dictated by the interaction between the protein and heme much of which has been deciphered through a variety of spectral probes and x-ray crystallography. Therefore, there are some specific questions that can be asked regarding the relationship between structure and function in heme proteins. The research in this proposal will focus on peroxidase. Recombinant systems have been developed for two peroxidases: cytochrome c and ascorbate peroxidase. Ascorbate peroxidase is the first and, to date, only eukaryotic heme enzyme that has been expressed as a holo-enzyme in E. coli. One goal of this proposal is to fully characterize the ascorbate peroxidase and then use mutagenesis to study its function. This will complement the ongoing work on cytochrome c peroxidase where mutagenesis will be used to probe the mechanism of intraprotein electron transfer. Since the substrate specificities of both peroxidases are markedly different and we have the crystal structures of both, we are in a good position to employ mutagenesis methods to decipher what structural features control substrate specificity.